Process for purifying viral substances and composition



P 1, 1964 J. CHARNEY- 3,147,185

PROCESS FOR PURIFYING VIRAL SUBSTANCES AND COMPOSITION Filed Sept. 10,1959 4 Sheets-Sheet l NUCLEIC ACID PRECIPITATION PROCEDURE FORPURIFICATION AND CONCENTRATION OF" POLIOVIRUS SCALE OF OPERATION TISSUECULTURE FLUID FILTRATE- loou TEns CONTAINING POLIOVIRUS STEP COOL TOBETWEEN |-|oc., 500 ml.

.1 ADD TO IOOjLq-l ml. NUCLEIC ACID I ADJUST TO pH 2-4, LET STAND ABOUTIB HOURS.

DRAW OFF CLEAR SUPERNATANT, A

CENTRIF UGE SEDIMENT, WASH SEDIMENT WITH PH 3.5 BUFFER.

OPTIONAL STEPS STEP DISSOLVE STEP 1 SEDIMENT m AQ. BUFFER, pH 4.5-9 500ml.

1: DIGEST SOLUTION wm-a (IIRIBONUCLEASE OR (2)R|Bo-uc| EAsE ANDDEOXYRIBONUCLEASE 2 (ACTIVATED WITH M9804) ABOUT IHR. AT ABOUT 37' C.

STEP DIGEST FURTH UR WITH .PURIFIED FICIN (ACTIVATED 500 ml.

III WITH CYSTEINE) ABOUT IHR. AT ABOUT 37C.

STEP DILUTE DIGESTION MIXTURE WITH 2 VOLS. H O, I500 ml.

III ADJUST TO ABOUT pH 3.5,

CENTRIF'UGE AT LOW SPEED.

DISCARD SUPERNATANT, WASH PELLET WITH ABOUT pH 3.5 BUFFER.

STEP DISSOLVE PELLET m AQ. BUFFER, pH 4.5 -9 40 ml.

11 CENTRIFUGE AT 40,000 RPM, ABOUT 5 HRS'.

DISCARD SUPERNATANT WASH PELLE'I' WITH Ao. BUFFER, pH 4.5,

DISSOLVE PELLET m AQuBUFFER, pH 4.5-9

CLARIFY BY CENTRIFUGATION,

DISSOLVE AND GLARIFY AGAIN As ABOVE,

DISCARD PELLET.

l CONCENTRATED, PURIFIED vmus SOLUTION.

INVENTOR JESSE CHARNEY FIG. I

p 1, 1964 J. CHARNEY 3,147,185

PROCESS FOR PURIFYING VIRAL SUBSTANCES AND COMPOSITION Filed Sept. 10,1959 4 Sheets-Sheet 2 OPTIOAL DENSITY O l l I l l I I r 200 20- 40 6O 80300 20 4O 6O WAVE LENGTH M/ POLIO V|RUS, l64mcg./m|.,CF/mcg.=3..7

(purified by process of exomple8 PURE POLIO VIRUS SPECTRUM FROM THE DATAOF SCHWERDT AND SCHAFFER CONGENTRATIONZ I64 mcg./ml., GF/mcg.= 3-5INVENTOR F l G. 2 JESSE CHARNEY PROCESS FOR PURIFYING VIRAL SUBSTANCESAND COMPOSITION Filed Sept. 10, 1959 Sept. 1., 1964 4 Sheets-Sheet 3ZDmFUMQW cum/mu IIITIIIHI INVENTOR JESSE CHARNEY ABSORBANCE (OPTICALDENSITY) J. CHARNEY Sept. 1, 1964 PROCESS FOR PURIFYING VIRAL SUBSTANCESAND COMPOSITION Filed Sept. 10, 1959 4 Sheets-Sheet 4 CNN 0 0 OPTICALDENSITY INVENTOR JESSE CHARN EY United States Patent 3,147,185 PRGCESSFUR PUREFYING VIRAL SUBSTANiIES AND CUMPU1ITION Jesse Charney, Lansdaie,Pa, assignor to Merck & $0., Inc, Rahway, N .17., a corporation of NewJersey Fiied Sept. 10, 1959, Ser. No. 839,543 7 Claims. ((11. 16i'78)This invention relates to an improved method for concentrating andpurifying viral substances. More particularly it relates to a process ofseparating, in highly concentrated and purified form, viral substancesfrom viral substance-containing fluids such as tissue culture fluids orVaccines made by the elimination of the infective property of tissueculture fluids by reaction with formaldehyde, long incubation, etc., andfurther relates to the use of pure concentrated viral substances in thepreparation of vaccines, and the vaccines thus produced.

The preparation of vaccines for use in preventing diseases of viralorigin, particularly poliomyelitis, has recently been simplified bygrowing the causative virus in tissue culture. The tissue culture mediamay be of several types, for example in growing poliomyelitis in monkeykidney tissue culture, the monkey kidneys are either minced or mincedand trypsinized. The work described herein was done on minced andtrypsinized monkey tissue culture fluids containing poliomyelitis virus(hereinafter referred to as poliovirus) but would be equally applicableto the minced tissue culture fluid with an even greater increase in thepurification factor achieved, or to fluids or filtrates obtained fromother tissue cultures.

At the end of the poliovirus growth period, tissue culture fluids areextremely dilute solutions of the desired viral substances contaminatedwith large amounts of impurities, many of them undesirable.

While methods are known which are used to concentrate and purify viralsubstances from tissue culture filtrates, these require that a largenumber of procedural steps be employed with the attendant loss in yieldsand opportunity for contamination. In addition they are tedious and timeconsuming. For example, the method described by Schwerdt & Schaffer,Annals of the New York Academy of Sciences, volume 61: article 4, page740 (1955), by which poliovirus was obtained in a pure state, prior toapplicants invention, involved (1) precipitation of the virus in thepresence of methanol, filtration of the virus so precipitated with theaid of a carrier (such as Celite), and elution of the filter-cake, donerepeatedly; (2) repeated emulsification with butanol to remove protein;(3) digestion with enzymes; (4) two cycles of ultracentrifugation; (5)final ultracentrifugation in a sucrose solution of graded density; and(6) removal of the sugar.

It can readily be seen that such complicated techniques applied to largequantities of dangerously infective virus fluids are not attractive fromthe point of view of commercial manufacture and that a method whichsimply and quickly results in an essentially pure virus product would beof great importance.

The disadvantages and hazards of the known methods for purifying andconcentrating viral substances are completely overcome by the method ofmy invention which provides a means for obtaining pure viral substancequickly and simply and in substantially quantitative yields from impurevirus-containing fluids or vaccine. The process of my invention shown inFIG. 1 of the accompanying draw- "ice ings, is based upon a newprinciple, namely that viral substances can be precipitatedquantitatively and in highly purified form from dilute or concentratedimpure viruscontaining solutions by adding nucleic acid and adjustingthe solution to a pH between about 2-4.

While the viral material obtained by this one step precipitation issufl'iciently purified and concentrated for use in preparing a vaccine,further purification can be effected by dissolving the precipitatedviral material in aqueous buffered solution at a pH range of about4.5-9, but preferably between pH 6.5-8.5, and then removing the residualnucleic acid and impurities which are primarily proteinaceous materialsby digestion of the solution with (1) ribonuclease or (2) ribonucleaseand deoxyribonuclease in the presence of magnesium ions (derived fromany soluble magnesium salt) which activate the deoxyribonuclease. Thedigestion is continued for about one hour at about 37 C. and purifiedficin (preferably activated by cysteine) is added and digestioncontinued for an additional hour at about 37 C. These enzymaticdigestions, without exerting a deleterious eflect upon the viralsubstance, convert the nucleic acid and proteinaceous materials to suchphysical and/ or chemical forms that they remain soluble duringsubsequent precipitation of the viral substance. At this stagesubstantially all of the nucleic acid has been digested but a tracegenerally remains which is adequate to reprecipitate the viral substanceupon adjusting the pH to between 2-4. The precipitated viral substancethen can be separated, for example by centrifugation, and the remainingpellet taken up in buttered solution, pH 4.5-9. If desired, the butteredsolution of the viral material can be ultracentrifuged and the pellet ofviral material taken up in a small quantity of buffered solution, pH4.5-9. If after the digestion with nuclease and ficin it is found thatthe viral substance does not precipitate upon adjusting the pH tobetween about pH 2-4, adjustment of the solution to a greater pH and theaddition of a very small quantity of nucleic acid will precipitate thedesired viral material upon subsequent reacidification.

The nucleic acid employed in the process of my invention can be of theribonucleic acid or deoxyribonucleic acid type (that is, plant or animalnucleic acid). That portion of nucleic acid which functions toprecipitate the viral substance is the undegraded (or highlypolymerized) nucleic acid. This can be obtained from commerciallyavailable nucleic acid by purification thereof by the method describedbelow or the crude nucleic acid can be used in larger quantity. Forexample, 1 gamma (mcg) per milliliter (ml.) of highly polymerizednucleic acid derived by purification of commercial yeast nucleic acid(Schwarz Laboratories, Inc., Mt. Vernon, New York), is effective inprecipitating poliomyelitis virus from its solution in crude, infectedmonkey kindey tissue culture fluid or other tissue culture fluids. Thisquantity (1 meg.) can be obtained from approximately 30 mcg. of thecrude acid. By adding the latter quantity of crude nucleic acid (30mcg.) the same effect is achieved without prior purification.

The advantages of the use of nucleic acid to eifect concentration ofviral substances from tissue culture fluid are manifold: (1) very smallquantities are required, 50-200 nag/liter of crude commercial product or0.5-2 mg./liter of purified product are suflicient to effect theprecipitation of the viral substance, higher concentrations can beemployed, if desired, although not usually necessary; (2) the nucleicacid is easily obtainable and inexpensive, especially if yeast nucleicacid is used; (3) the precipitate of nucleic acid and virus is readilysoluble in neutral aqueous solutions; (4) successive precipitations canbe carried out without loss of viral matter; (5) and the nucleic acidcan be separated from the virus material by digestion with a nucleasesuch as ribonuclease or a mixture of ribonuclease and deoxyribonuclease.The split products of the nucleic acid digestion then can be eliminatedby dialysis, ultracentrifugation or reprecipitation of viral material bythe addition of an alcohol, for example ethanol or methanol, thedigestion products remaining in the supernatant.

Subsequent digestion with purified ficin or other proteolytic enzymes,for example, papain, removes proteinaceous impurities. When purifiedficin is employed it is advantageously activated by the addition ofcysteine which is usually employed in a ratio of parts by weight ofcysteine to each part of ficin.

The precipitation of viral material at pH 2-4 in the presence of nucleicacid advantageously is conducted at a temperature between about 110 C.While the best pH range lies between 2.040, the acidity can be increasedor decreased to between about pH 1.5 to about pH 4.5 and the virussuccessfully precipitated provided, when the acidity is increased toabout pH 1.5, the step be carried out as quickly as possible to avoidacid degradation of the virus or viral material. The acid employed toadjust the pH can be a mineral acid such as hydrochloric, sulfuric orphosphoric, or an organic acid such as citric, oxalic or other organicacid. Hydrochloric acid is as effective as any acid and as it is areadily available laboratory reagent it was employed in the processdescribed herein as a matter of convenience.

In general, the product obtained at this point represents substantiallypure poliovirus, the poliovirus being 80- 100% pure. The major impurity,if any, at this point, is the material known to the art as C componentwhich is related to the poliovirus, and is, in fact, the outer proteinenvelope of the virus particle which lacks the central core of nucleicacid. Inactivation of the virus thus obtained by methods known to theart, will result in substantially pure poliovirus antigen. However, ifthe process is performed using an imperfectly filtered tissue culturefluid or without due care, the product obtained will not besubstantially pure. Nevertheless, even under these conditions, theproduct will contain poliovirus as a major component with respect to thetotal protein in the concentrate, and inactivation of this product bymethods known to the art will yield a vaccine containing poliovirusantigen as the major portion of the total protein in the vaccine. Thevaccine so obtained is fully effective for immunization againstpoliomyelitis disease and could be sold as a marketable product.

The preparation of purified viral material by the method of my inventionis of considerable commercial importance because for the first time itis now possible to accomplish its concentration and purification inconsiderably less time than was needed when employing known procedures,and the virus is obtained in substantially quantitative yields. Thepurified and concentrated virus which is obtained by the process of myinvention if of commercial importance because it can be converted to apurified vaccine largely or wholly freed from undesirable matter. Suchvaccine prepared from the purified and concentrated virus obtained bythe process of my invention has many advantages over vaccines heretoforeprepared, such as (1) superior safety because the reproducibility ofextinction of infectivity can be guaranteed by the use of knownconcentrations of pure virus; (2) absence of undesirable foreignprotein; (3) constant, known dosages can be administered in any chosenvolume; and (4) for the first time it is possible to incorporate thedesired amount of the pure inactivated virus into vaccines containingother antigens 4 all of which then can be administered simultaneously ina feasible single dosage form.

The process of my invention is more fully described in the followingexamples which illustrate not only the reproducibility of the processbut also feasible ranges in which the process is operable.

The examples also illustrate, as another feature of my invention, thepreparation of an effective vaccine prepared by inactivation ofpurified, concentrated poliovirus.

PREPARATION OF REAGENTS EMPLOYED IN PROCESSES DESCRIBED IN EXAMPLESPurification 0] Crude Yeast Nucleic Acid Commercially available yeastnucleic acid (10 grams), [obtained from Schwarz Laboratories, Inc., Mt.Vernon, New York, Lot No. NH5704, hereinafter referred to as yeastnucleic acid (Schwarz)], were dissolved in one liter of 1% phosphatebuffer (pH 7.0) containing 2% sodium chloride. The pH of this solutionwas adjusted with 10 N hydrochloric acid to pH 3.0, causing formation ofa precipitate which was allowed to settle. The supernatant solution wasdecanted and the precipitate was redissolved in ml. of 1% phosphatebuffer (pH 7.0) containing 2% sodium chloride. The pH of this solutionwas adjusted to pH 3.0 with 10 N hydrochloric acid and the resultingsuspension centrifuged for 15 minutes at 3000 rpm. (revolutions perminute) at 2 C. The supernatant solution was discarded and theprecipitate was dissolved in 50 ml. of 1% phosphate buffer (pH 7.0)containing 2% sodium chloride. Spectrophotometric examination showed theconcentration of purified yeast nucleic acid in the solution to be 300Meg/ml. This was a convenient form for use of this material in thepreparation of purified, concentrated viral substance.

The yield of purified yeast nucleic acid obtained in this purificationprocedure (15 mg. from 10 gm. of starting material) was so low thatexperiments were conducted using the commercially available yeastnucleic acid. The crude material proved satisfactory (see for example,Example 4 below) when used at concentrations forty to one hundred foldthat of the purified nucleic acid.

Purification of F icin Ficin (Merck & Co., Inc., Rahway, New Jersey), 2grams, was suspended in 20 ml. of acetate buffer (pH 5.0, 0.01 ionic).The suspension was stirred without foaming for one hour resulting in ahazy solution. This solution was centrifuged for 15 minutes at 3000r.p.m. at 10 C. Two grams of Celite 503, a diatomaceous filter aidmanufactured by the Johns-Manville Company, were added to thesupernatant solution which then was filtered through paper by gravity.The filtrate was cooled to 1-5 C. and methanol, which had been cooled to20 C. added with stirring to a final concentration of 40% (v./v., thatis, volume per volume). The mixture was kept at -l0 C. overnight andthen centrifuged, the supernatant discarded and the precipitateextracted with three 5 ml. portions of distilled water, each extractionmixture being centrifuged at 2 C. The pooled extracts were stored at 5C. for one week then centrifuged to remove the precipitate which formedand the supernatant solution lyophilized. The lyophilized product, whichis completely soluble in water, contains 70% of the initial activity.

ASSAY METHOD In evaluating the efficacy of a purification procedure asapplied to viruses, the need for a simple, accurate, precise and promptassay becomes immediately apparent. In the case of poliovirus, theprincipal methods of assay in use are based on (a) the metabolicinhibition (color) test [also known as infectivity (color) test]described by Salk, Youngner and Ward, American Journal of Hygiene,

60: 214 (1954); (b) the plaque forming test described by Hsiung andMelnick, Virology 1: 533 (1955); and (c) the complement fixation method(CF) described by Osler, Strauss and Mayer, American Journal ofSyphilis, Gonorrhea and Venereal Diseases, 36: 140 (1962). The last ofthese methods measures total antigenic mass present while the first twomethods measure only that minor portion of the antigen mass which isinfective. All three methods have been used in this work with goodcorrelation, as shown in Table A below. For reasons of convenience,simplicity and speed it was decided to place chief reliance on thecomplement fixation (CF) method and that method of assay is used in theexamples illustrating the novel process of this invention.

TABLE A.CORRELATION OF ASSAY MEASURES USED TO FOLLOW PURIFICATION WITHPOLIOVIRUS 1 C13 means complement fixing units, assay according tomethod described by Osler, et a1 above.

Crude Viral Starting Material Unless otherwise noted the tissue culturefiltrates employed in the following examples were obtained by growingthe virus on monkey kidney cells and harvesting by known methods.

EXAMPLE 1 (This example shows that the use of 5 meg/ml. of purifiedyeast nucleic acid in the process of this invention results inquantitative concentration and recovery of viral substance) A tissueculture filtrate containing Type I (Mahoney) poliovirus was found tocontain a total of 300 complement fixing (CF) units per 100 ml. To a 100ml. aliquot of this tissue culture filtrate, 1.67 ml. of a 300 meg/ml.solution of yeast nucleic acid (Schwarz) purified as described above,was added giving an overall concentration of 5 meg/ml. of purified yeastnucleic acid. This solution was cooled to 5 C., adjusted to pH 2.5 with1 N hydrochloric acid, centrifuged 15 minutes at 3000 r.p.m. at 2 C. Thesupernatant was decanted and the pellet dissolved in 5 ml. of 1%phosphate buffer (pH 7.0) containing 2% sodium chloride. The supernatantwas found to contain no CF units while the solution of the pelletcontained a total of 320 CF units indicating a volume concentration oftwenty-fold with no loss of activity.

As a control, another 100 ml. aliquot of this solution was cooled to 5C., adjusted to pH 2.5 with 1 N hydrochloric acid and centrifuged 15minutes at 3000 r.p.m. at 2 C. The supernatant was decanted and thepellet dissolved in 5 ml. of 1% phosphate buffer (pH 7.0) containing 2%sodium chloride. The supernatant was found to contain a total of 200 CFunits; the solution of the pellet contained none, indicating that noconcentration was achieved.

EXAMPLE 2 (This example shows that the minimum concentration of purifiedyeast nucleic acid required for substantially quantitative precipitationof poliovirus from tissue culture filtrates is more than 1.25 meg/ml.and probably less than 2.5 mega/ml.)

To four 100 m1. aliquots of a tissue culture filtrate containing TypeIII (Saukett) poliovirus and having a CF activity of 250 units/100 ml.there was added decreasing amounts (1.67, 0.84, 0.42, 0.21 ml.) of the300 meg/ml. solution of yeast nucleic acid (Schwarz), purified by theprocess described above, to give final solution concentrations ofpurified yeast nucleic acid of 5 mcg./ml., 2.5 mcg./ml., 1.25 meg/ml.and 0.625 meg/ml. respectively.

All the aliquots were cooled to 5 C., adjusted to pH 2.5 with 1 Nhydrochloric acid, centrifuged 15 minutes at 3000 r.p.m. at 2 C. Thesupernatants were discarded and each of the pellets dissolved separatelyin 5 ml. of 1% phosphate buffer (pH 7.0) containing 2% sodium chloride.The solutions of the pellets were assayed with the results shown inTable B below:

(This example shows that poliovirus precipitated with purified yeastnucleic acid is stable under the conditions tested (pH 2.5, 5 C.) for atleast 21 hours) To a 500 ml. portion of the same tissue culture filtratecontaining Type III (Saukett) poliovirus used in Example 2, whichcontained 250 CF units per ml., 8.33 ml. of a 300 mcg./ ml. solution ofyeast nucleic acid (Schwarz), purified as described above, were added togive a final concentration of 5 meg/ml. The solution was cooled to 5 C.and then adjusted with 1 N hydrochloric acid to pH 2.5, causingformation of a precipitate, and the mixture then was stored at 5 C. Atthe time intervals stated in Table C below, the mixture was stirred tosuspend the precipitated material and a 100 m1. aliquot removed. Eachaliquot was centrifuged 15 minutes at 3000 r.p.m. at 2 C. Thesupernatant was discarded and the pellet dissolved in 5 ml. of 1%phosphate buffer (pH 7.0) containing 2% sodium chloride. The solutionsof the pellets were assayed by the complement fixation test with theresults shown below:

(This example shows that commercially available crude yeast nucleic acidcan be used to precipitate poliovirus from tissue culture filtrates in asubstantially quantitative yield and demonstrates that the minimumconcentration required to achieve this yield is greater than 25 meg/ml.and probably less than 50 meg/ml.)

To four 100 ml. aliquots of the same tissue culture filtrate [Type Ill(Saukett) poliovirus] used in Example 2, having an activity of 250 CFunits per 100 ml., was added in decreasing quantities (1, 0.5, 0.25 and0.125 ml.) of a 20 mg./ ml. solution of crude yeast nucleic acid(Schwarz) thus providing final yeast nucleic acid concentrations givenin Table D. All the aliquots were cooled to 5 C., adjusted to pH 2.5with 1 N hydrochloric acid and centrifuged 15 minutes at 3000 r.p.m. at2 C. The supernatants were discarded and each pellet dissolvedseparately in 5 ml. of 1% phosphate butter (pH 7.0) containing 2% sodiumchloride. The solutions of the pellets were assayed with the resultsshown in Table D below:

(This example shows that the pH of the nucleic acid precipitation can bemore alkaline than pH 2.5, at least up to pH 3.5)

Ten ml. of a 20 mg./ml. solution of yeast nucleic acid (Schwarz) wereadded to one liter of a tissue culture filtrate containing Type III(Saukett) poliovirus, having an activity of 4000 CF units per liter, togive a concentration of yeast nucleic acid of 200 meg/ml. This solutionwas cooled to 5 C., adjusted to pH 3.5 with 1 N hydrochloric acid andallowed to settle at 5 C. for 18 hours. Approximately 900 ml. of clearsupernatant were decanted and the remaining sediment centrifuged 15minutes at 3000 r.p.m. at 2 C. The clear supernatant was discarded andthe pellet dissolved in ml. of 1% phosphate butter (pH 7.0) containing2% sodium chloride. The solution of the pellet was assayed and found tocontain a total of 4000 CF units, indicating 100% recovery of theactivity initially present in the tissue culture filtrate, and in 1/100of the original volume.

EXAMPLE 6 (This example shows that incubation of a concentrated solution of poliovirus with 100 meg/ml. of purified ficin is not destructiveof the infectivity or complement fixation properties of the virus) Fiveml. of a 20 mg./ml. solution of yeast nucleic acid (Schwarz) were addedto one liter of a tissue culture filtrate containing Type III (Saukett)poliovirus to give a concentration of 100 meg/ml. The solution wascooled to 5 C. and adjusted to pH 2.5 with 1 N hydrochloric acid andallowed to settle at 5 C. for 18 hours. The clear supernatant wasdecanted and the sediment centrifuged minutes at 3000 r.p.m. at 2 C. Thesupernatant was discarded and the pellet dissolved in 10 ml. (1/100 theoriginal volume) of 1% phosphate buffer (pH 7.0) containing 2% sodiumchloride. A portion of this solution was assayed for infectivity andcomplement fixation with the results shown in Table E, item 1) below.The remainder of the solution was incubated for one hour at 37 C. with 1mg. of purified ficin prepared by the method described above plus 10mgs. of cysteine (to activate the ficin) dissolved in 1 ml. of water.After this incubation the solution was assayed for infectivity and CFwith the results shown in the table, item (2) below:

EXAIVIPLE 7 (This example describes removing the nucleic acid andresidual proteins remaining in the virus pellet by digestion withribonuclease and further digestion with ficin) Tissue culture filtratecontaining Type III (Saukett) poliovirus, one liter, containing a totalof 4000 CF units was cooled to 5 C. Yeast nucleic acid (Schwarz), 10 ml.of a 20 mg./ml. solution of crude nucleic acid (Schwarz) to give a finalsolution concentration of 200 mcg./ml. of nucleic acid, was added andthe H adjusted to 3.5 with 1 N hydrochloric acid. The solution wasallowed to stand at 5 C. for 18 hours, the supernatant then wasdiscarded and the sediment centrifuged 15 minutes at 3000 r.p.m. at 2 C.The supernatant was discarded and the pellet dissolved in 10 ml. of a 1%phosphate butler (pH 7.0) containing 2% sodium chloride. Upon assay thissolution was shown to contain a total of 4000 CF units. Thisconcentrated solution of poliovirus was digested with ribonuclease (10meg/ml.) for one hour at 37 C. An assay of the solution followingdigestion with ribonuclease showed it to contain a total of 3600 CFunits. Purified ficin n1cg./ml., prepared as described above) and 1rug/ml. of cysteine was added to the digestion mixture and digestioncontinued for an additional hour at 37 C. Assay of the solutionfollowing digestion with ficin showed it to contain a total of 2800 CFunits. The solution then was cooled to 5 C., diluted with 1.5 volumes ofcold distilled water and the pH adjusted to 2.5 with 1 N hydrochloricacid and centrifuged for 15 minutes at 3000 r.p.m. at 2 C. Thesupernatant was discarded and the pellet dissolved in 11.5 ml. of 1%phosphate buffer (pH 7.0) containing 2% sodium chloride. An assay ofthis solution showed it contained a total of 2800 CF units.

EXAMPLE 8 (This example describes preparation of pure poliovirus bynucleic acid precipitation at pH 3.5, followed by digestion withribonuclease then with purified ficin and reprecipitation at pH 2.5followed by ultracentrifugation to yield pure poliovirus) Tissue culturefiltrate containing Type III (Saukett) poliovirus, 2 liters, containinga total of 7000 CF units was cooled to 5 C. Yeast nucleic acid(Schwarz), 10 ml. of a 20 mg/ml. solution of yeast nucleic acid(Schwarz) to give a final solution concentration of 100 meg/ml. ofnucleic acid, was added and the pH adjusted to 3.5 with 1 N hydrochloricacid. The solution was allowed to stand at 5 C. for 18 hours, thesupernatant then was discarded and the sediment centrifuged 15 minutesat 3000 r.p.m. at 2 C. The supernatant was discarded and the pelletdissolved in 10 ml. of a 1% phosphate butter (pH 7.0) containing 2%sodium chloride. Upon assay this solution was shown to contain a totalof 6400 CF units. This concentrated solution of poliovirus was digestedwith ribonuclease (10 meg/ml.) for one hour at 37 C. An assay of thesolution following digestion with ribonuclease showed it to contain atotal of 4800 CF units. Purified ficin (100 mcg./ml., prepared asdescribed above) and cysteine (1mg./ml.) were added to the above mixtureand digestion continued an additional hour at 37 C. Assay of thesolution following digestion with ficin showed it to contain a total of400 CF units. The solution then was cooled to 5 C., diluted with 1.5volumes of cold distilled water, the pH adjusted to 2.5 with 1 Nhydrochloric acid and centrifuged for 15 minutes at 3000 r.p.m. at 2 C.The supernatant was discarded and the pellet dissolved in 11.5 ml. of 1%phosphate buffer (pH 7.0) containing 2% sodium chloride. This solutionassayed a total of 4000 CF units. The thus obtained solution wascentrifuged three hours at 40,000 r.p.m. at 2 C. The supernatant wasdiscarded, the pellet dissolved in 5 ml. of 1% phosphate butler (pH 7.0)containing 2% sodium chloride, again centrifuged for 10 minutes at10,000 r.p.m. at 2 C. and the pellet thus obtained dissolved in 10 ml.of 1% phosphate butter (pH 7.0) containing 2% sodium chloride. Thissolution assayed 3500 CF units.

Table F below summarizes the testing data in the above example at theend of each step of the process described.

TAB LE F Volume Total OF Percent Nature of sample ml) units initialactivity Original tissue culture filtrate 2, 000 7, 000 100 Supernatantfrom pH 3.5 precipitation- 2, 000 Solution of pH 3.5 precipitate 10 6,400 91 Above solution after digestion with ribonuclease 10 4, 800 69Above solution after digestion with purified ficin 1O 4, 000 57Supernatant from pH 2.5 precipitate. 25 5OO 7 Solution of pH 2.5precipitate l1. 5 4, 000 57 Ultracentrituged supernatant 11. 5 550 8Solution of ultracentrifuged pellet 3, 500 50 The purity of thepoliovirus obtained by the process described in this example wasestablished by a comparison of its ultra-violet spectrum with thespectrum published by Schwerdt and Schaifer, Annals of the New YorkAcademy of Science, 61: 740 (1955 The spectrum for the pure poliovirusprepared by the process of this invention and that prepared by Schwerdtand Schatfer is shown in FIG. 2 of the accompanying drawings. Acomparison of these spectra demonstrates that the spectrums are thesame, with a maximum at Wave length 260 mu and a minimum at wave length241 mu.

EXAMPLE 9 (This example describes removing the nucleic acid and residualproteins remaining in the virus pellet by digestion with a mixture ofribonuclease and deoxyribonuclease and further digestion with ficin)Tissue culture filtrate containing Type III (Saukett) poliovirus, 500ml., containing a total of 1750 CF units, was cooled to 5 C. Yeastnucleic acid (Schwarz), 5 ml. of a mg./ml. solution of yeast nucleicacid (Schwarz) to give a final solution concentration of 200 mcg./ ml.of nucleic acid, was added and the pH adjusted to 3.5 with 1 Nhydrochloric acid. The solution was allowed to stand at 5 C. for 20hours, the supernatant then was discarded and the sediment centrifuged15 minutes at 3000 rpm. at 2 C. The supernatant was discarded and thepellet dissolved in 10 ml. of 1% phosphate buffer (pH 7.0) containing 2%sodium chloride. Upon assay this solution was shown to contain a totalof 2000 CF units. This concentrated solution of poliovirus was digestedfor one hour at 37 C. with a mixture of ribonuclease (10 mcg./ml.),deoxyribonuclease (10 meg/ml.) and magnesium sulfate (MgSO -7H O, 10mg./ml.). An assay of the solution following digestion with a mixture ofribonuclease and deoxyribonuclease showed it to contain a total of 1600CF units. Purified ficin mcg./1nl., prepared as described above) andcysteine (250 meg/ml.) were added to the above mixture and digestioncontinued an additional hour at 37 C. Assay of the solution followingdigestion with ficin showed it to contain a total of 2400 CF units. Thesolution then was cooled to 5 C., diluted with 2 volumes of colddistilled water, the pH adjusted to 2.5 with 1 N hydrochloric acid andthen centrifuged for 15 minutes at 3000 rpm. at 2 C. The supernatant wasdiscarded and the pellet dissolved in 10 ml. of 1% phosphate buffer (pH7.0) containing 2% sodium chloride. Assay of this solution showed it tocontain 2660 CF units.

EXA PLE 10 (This example shows that the nucleic acid precipitationmethod of this invention can be used to concentrate virus grown on cellsother than monkey kidney cells) Type III (Saukett) virus was grown onrhesus testicular cells by conventional methods. When the cultures wereharvested, the cellular debris was removed by centrifugation. An aliquotof the clear tissue culture fluid was dialyzed against 0.85% sodiumchloride solution to permit its being measured in the complementfixation test, sample MT-61 Table G below, (undialyzed material couldnot be used because of color interference). To another 100 ml. aliquotof the clear virus containing fluid, 1 ml. of a 20 mg./ml. solution ofyeast nucleic acid (Schwarz) was added, thus providing a finalconcentration of 200 mcg./ ml. of yeast nucleic acid (Schwarz). Thesolution was cooled to 5 C., adjusted to pH 2.5 with 1 N hydrochloricacid, centrifuged 15 minutes at 3000 rpm. at 2 C. The supernatant wasdiscarded and the pellet dissolved in 10 ml. (V10 original volume) of 1%phosphate bufier (pl-I 7.0) containing 2% sodium chloride. The solutionof the pellet was submitted for CF testing with the results shown inTable G below, sample MT61C. Two additional aliquots were treatedidentically as described above and assayed with the results given in thefollowing table for samples MT-62 and MT-62-C.

EXAMPLE 11 (This example shows that the nucleic acid precipitationmethod of this invention preferentially concentrates the viral antigenrather than anticomplementary material and thereby permits use of the CFtest in these studies) Some virus-containing fluids contain so muchanti-complementary material (see, for example Boyd, W. C., Fundamentalsof Immunology, 2nd edition (1947), page 298; Interscience Publishers,Inc, New York, N.Y.), that, even after dialysis, they can not bemeasured in the complement fixation test. This is a serious handicap inthose studies whose purpose is the growth of increased amounts of viralantigen. Type II (MEF-l) poliovirus was grown on rhesus testicularcells. When the culture was harvested, the cellular debris was removedby centrifugation. An aliquot of the clear virus-containing supernatantwas dialyzed against 0.85% sodium chloride solution for measurement inthe CF test. To another ml. aliquot of this clear virus-containingsupernatant there was added 1.67 ml., of the 300 meg/ml. solution ofyeast nucleic acid (Schwarz) purified by the process described above togive a final concentration of 5 mcg./ ml. of the purified yeast nucleicacid. The virus-containing solution then was cooled to 5 C., adjusted topH 2.5 with 1 N hydrochloric acid and centrifuged 15 minutes at 3000rpm. at 2 C. The supernatant was discarded and the pellet dissolved in10 ml. original volume) of 1% phosphate buffer (pH 7.0) containing 2%sodium chloride. The dialyzed original fluid [Table H, item (1)] and the10-fold concentrate resulting from solution of the pellet [Table H, item(2)] were assayed for infectivity by the color and plaque tests and forCF with the results shown in Table H be ow:

1 Anticomplcmentary.

The data in Table H indicate that: (1) nucleic acid can be used toprecipitate poliovirus from viral fluids whose origin was other thanmonkey kidney cells: (2) the concentrations achieved by the nucleic acidprecipitation method are quantitative; (3) the data obtained byinfectivity measurements as well as CF measurements establish theeffectiveness of the nucleic acid precipitation method; and (4) thenucleic acid precipitation method concentrates viral antigen inpreference to anticomplementary matter permitting use of the CF test inviral growth studies. This is an analytical aid of considerableimportance because of the greater convenience and precision it confers.

EXAMPLE 12 (This example demonstrates that the yeast nucleic acidprecipitation method of this invention can be used to concentratenon-infective antigen from vaccines, thus provid ing a method forreadily measuring antigen content in those vaccines containinginsufficient concentration of antigen to be measured by the complementfixation test) Commercially prepared poliomyelitis vaccine (Merck Sharp& Dohme, Lot No. 29252), 900 ml., was used. This gave negative resultson CF test when tested after appropriate dialysis to remove color. Yeastnucleic acid (Schwarz), 9 ml., in the form of a 20 mg./ml. solution, wasadded to give a final solution concentration of 200 meg/ml. The solutionwas cooled to C. then adjusted to pH 2.5 with l N hydrochloric acid andset aside at 5 C. for 18 hours. The clear supernatant was decanted andthe remaining sediment centrifuged for 15 minutes at 3000 r.p.m. at 2 C.The supernatant was discarded and the pellet dissolved in 10 ml. of0.85% sodium chloride solution. This solution was tested for infectivity(by color test) and also tested by complement fixation. No infectivitywas found. The following CF values were obtained against Type I35(total), indicating 0.39 per ml. in the original Type IIl2.5 (total),indicating 0.14 per ml. in the original Type III-10 (total), indicating0.11 per ml. in the original EXAMPLE 13 (This example shows that thenucleic acid precipitation meth od can be used to concentrate infectivevirus present in a vaccine from a level undetectable by the color testto one which is readily detectable. It offers a means of making thesafety testing of vaccines more sensitive and less laborious, inasmuchas the entire product from several liters of vaccine can he tested in afew small tissue culture flasks) To one liter of the vaccine at 5 C.,described in Example 12, there was added 10 ml. of a 20 mg./ml. solutionof yeast nucleic acid (Schwarz) to give a final concentration of 200meg/ml. To this solution there was added 1,000 tissue culture infectivedoses (TCID of Type III (Saukett) poliovirus yielding a virusconcentration of 10, i.e. 1.0, TCID /ml. The pH of this solution wasadjusted to 2.5 with concentrated hydrochloric acid. It was allowed tostand 17-18 hours at 5 C.

The clear supernatant was decanted and the remaining sedimentcentrifuged minutes at 3000 r.p.m. at 2 C.

The supernatant was discarded and the pellet dissolved in 10 ml. of thenutrient solution used in the infectivity (color) test. This solutionwas tested for infectivity and complement fixation. Infectivity titerwas 10 i.e.

17.8, TClD /ml; CF values were identical with those reported in Example12.

EXAMPLE 14 (This example describes the preparation of efiective vaccinesprepared by the formaldehyde inactivation of two different Type Istrains of pure poliovirus) A batch of monkey kidney cells, monolayercultures, were prepared. Half of the flasks were seeded with Mahoneypoliovirus and the remaining half with Parker poliovirus. From theinfected fluids obtained upon harvesting the cultures, pure virus ofeach type was isolated having an ultra-violet absorption spectrumidentical with that given for the pure virus prepared by the method ofExample 8. Each pure virus was dissolved in 1% phosphate buffer, pH 7.0,at a concentration of 20 meg/ml. These solutions of virus wereinactivated by adding formaldehyde (l:4000 formalin) to a concentrationof 92.5 mcg./ml., placing in vials which are completely filled withsolution and sealed, wrapped in aluminum foil to exclude light, andincubating at 37 C. for 168 hours. The contents of the vials then werediluted 1:5 with 0.85% sodium chloride solution, thus providing aconcentration of l mcg. of inactivated virus per ml. This mixture ishereinafter referred to as the vaccine. A small portion of each vaccinewas dialyzed at 5 C. about 18 hours against 0.85% sodium chloridesolution and then 24 hours against the nutrient solution used in theinfectivity (color) test. The results of the infectivity test of eachvaccine were negative. These vaccines were tested for effectiveness inmonkeys by the procedure established by the National Institutes ofHealth (NIH) and described in the Federal Register, volume 21, No. 128,page 4924, paragraph 73.103, July 3, 1956, with the exception that, foreach vaccine, dosages of 1 ml. of the monovalent vaccine were employedand 8 monkeys per test were used, all 8 monkeys surviving the test ineach case. The geometric mean ratio calculated from the data thusobtained for the vaccine prepared from pure Mahoney poliovirus was 0.76,and that from the vaccine prepared from pure Parker poliovirus was 0.54;NIH minimum ratio requirement, 0.29.

EXAMPLE 15 (This example gives the physical and chemical properties ofthe non-infective poliomyelitis antigen) The physical and chemicalproperties of the non-infective antigen prepared by inactivation ofpoliovirus, purified by the novel method of my invention, have beendetermined on a representative sample of antigen by known analyticalmethods. The data thus obtained characterize the antigen within theprecision of these analytical procedures with high molecular weightprotein substances. The antigen was prepared by the inactivation ofpoliovirus, substantially completely free from non-poliovirus protein,by essentially the same method described in Example 14.

The elemental analysis, infrared and ultraviolet spectra provide usefuldata for the characterization of the noninfective antigen.

The carbon and hydrogen analyses were made by the Pregl combustionmethod modified by use of tungstic oxide as catalyst, described inOrganic Quantitative Microanalysis, Niederl and Niederl (1942), pages101- 150. The nitrogen determination was by the method of Johnsondescribed in Monometric Techniques," 3rd edition (1957), page 238,published by Burgess Publishing Company. Analysis for phosphorus was bythe method of Fiske and Subba Row, Methods in Enzymology, vol. III(1957), page 843, Academic Press Inc. Sulfur was determined by drycombustion according to the method of Steyermark, Quantitative OrganicMicroanalysis (1951), pages 167-177, followed by volumetric titration bymethod of Ogg, Willits and Cooper, described in their paper, Volumetricdetermination of small amounts of soluble sulfates, in AnalyticalChemistry, vol. 20 (1948), page 83. The analytical sample used was alyophilized, non-infective poliomyelitis antigen which was dried toconstant weight over P 0 at room temperature. The non-infective polioantigen, analyzed by the above procedures and apparatus, was found tocontain the following elements in the proportions specified, calculatedon a moisture-free basis at its isoelectric point:

1 By difference.

The infrared spectrum of the antigen, shown in FIG. 3 and FIG. 4 of theattached drawings, was determined in a Perkin & Elmer Corp. infraredspectrophotometer Model 21 and Model 137 KBr prism spectrophotometer.

When mixed in a KBr pellet in the ratio of approximately 1 part ofantigen to 350 parts KBr, the antigen shows a number of characteristicmaxima in the infrared region, the more significant of which are thefollowing:

Wave Percent length transmis- Absorbance sion 1 Region. 2 Weak band.

3 Broad and weak.

The following data were obtained:

Millimicra Maximum absorption 259-261 Minimum absorption 241 maximum 280(protein peak) density (A 260) 21%,;825

Ratio =1.63

The ratio given above bears upon the freedom from foreign protein. Allproteins have an absorption peak at 280 millimicra. In a product havingan absorption at a different wave length but likely to contain foreignprotein, the ratio of absorption at the maximum to absorption at 280 hassignificance.

In addition to the above data which is useful to characterize thenon-infective poliomyelitis antigen, the values for particle size,particle count per unit weight, sedimentation coefficient, diffusioncoefficient and molecular weight, together with data bearing onbiological specificity, especially specific complement fixing abilityand immunizing capacity are more pertinent for the characterization ofthe poliomyelitis antigen.

The size of the non-infective antigen particle was found to be 28millimicrons, and the number of particles per microgram of antigen wasfound to be 934x when determined by the methods described by C. E. Hall,Introduction to Electron Microscopy (1953), pages 369-373 (McGraw-HillBook Co.). The data was obtained on a Phillips Model 100A ElectronMicroscope.

Sedimentation studies of the antigen were performed on a Spinco Model Banalytical ultracentrifuge using an A-N rotor. The methods used forcomputing the sedimentation coefficient, diffusion coefficient, andmolecular weight are described by H. K. Schachman, Methods inEnzymology, vol. IV (1957), pages 32-77 and 102-103.

The data obtained for the antigen by these methods are:

Sedimentation coefiicient D 155 l0 per sec.

Diffusion coeflicient D 1.89 10- per cm.

per sec.

Molecular weight 6,500,000.

Assay by complement fixation of each of the three types of antigen, bythe method hereinbefore described, gave the following results.

Units/meg.

2.5 (positive) 0.25 (negative) 0.25 (negative) 5.7 (positive) 0.5(negative) 0.5 (negative) A vaccine containing non-infective polioantigen, prepared by inactivating poliovirus purified and concentratedby the process of this invention was administered to 35 triple-negativechildren, i.e., children who by neutralization test were demonstrated topossess no immunity against any of the three types of polio. The vaccinecontained 3.0 mcg. per 0.5 ml. Type I, 0.6 mcg. per 0.5 ml. Type II, and1.0 mcg. per 0.5 ml. Type III, and was administered in two doses of 0.5ml. each 1 month apart. One month following the second dose, a sample ofblood was taken to determine the titre of antibody produced. Each childwas found to have a positive titre to each of the types of poliovirus asshown in the following table.

TABLE I Conversion vs. type No. of triple neg. children II III ParkerMahoney This application is a continuation-in-part of my copending US.patent application, Serial No. 660,170, filed May 20, 1957, nowabandoned.

While the invention has been illustrated by certain specific exampleswhich illustrate the purification and concentration of poliovirus, thepreparation of a noninfective poliomyelitis antigen therefrom and thepreparation of a vaccine suitable for administering the antigen toimmunize against poliomyelitis, it is to be understood thatmodifications in the procedural steps and in the reagents used can bemade within the framework of the general disclosure and of the appendedclaims.

What is claimed is:

1. A process for purifying and concentrating poliomyelitis viralsubstances from poliomyelitis viral substance-containing fluids,comprising adding nucleic acid to the poliomyelitis viralsubstance-containing fluid and adjusting the pH to between 2-4 andrecovering the precipitated poliomyelitis viral substance.

2. A process for purifying and concentrating poliomyelitis viralsubstances from poliomyelitis viral substance-containing fluids,comprising adding nucleic acid to the poliomyelitis viralsubstance-containing fluid and adjusting the pH to 3.5 and recoveringthe precipitated poliomyelitis viral substance.

3. A process for purifying and concentrating poliomyelitis virus frompoliomyelitis virus-containing tissue culture filtrates comprisingadding nucleic acid to the poliomyelitis virus-containing tissue culturefiltrate and adjusting the pH to between 2-4 and recovering theprecipitated poliomyelitis virus.

4. A process for purifying and concentrating poliomyelitis virus frompoliomyelitis virus-containing tissue culture filtrates comprisingadding nucleic acid to the poliomyelitis virus-containing tissue culturefiltrate and 15 adjusting the pH to 3.5 and recovering the precipitatedpoliomyelitis virus.

5. A process for purifying and concentrating poliomyelitis virus frompoliornyelitis virus-containing tissue culture filtrates comprisingadding yeast nucleic acid to the poliomyelitis virus-containing tissueculture filtrate and adjusting the pH to between 2-4 and recovering theprecipitated poliomyelitis virus.

6. A process for purifying and concentrating poliornyelitis virus frompoliornyelitis virus-containing tissue culture filtrates comprisingadding yeast nucleic acid in a quantity sutficient to provide theequivalent of at least about 2.5 meg/ml. of purified nucleic acid to thepoliornyelitis virus-containing tissue culture filtrate and adjustingthe pH to 3.5 and recovering the precipitated poliomyelitis virus.

7. A process for preparing concentrated, pure poliomyelitis virus frompoliomyelitis virus-containing tissue culture filtrates comprisingadding nucleic acid to the poliornyelitis virus-containing tissueculture filtrate and adjusting the pH to between 2-4, recovering theprecipitated poliornyelitis virus and digesting a buffered solution ofthe virus with ribonuclease and then with a. J purified ficin activatedby cysteine, then adjusting the pH to between 2-4 and recovering purepoliomyelitis virus.

References Cited in the file of this patent UNITED STATES PATENTSValentine July 26, 1960 Cox Dec. 27, 1960 OTHER REFERENCES Schwerdt andSchatfer: Annals of the New York Academy of Sciences, pp. 740753, 1955.

Schaffer: Advances in Virus Research, 1959, pp. 181 and 203.

Rivers et al.: Viral and Rickettsial Infections of Man, page 37, pub.1959, J. B. Lippincott Co., Philadelphia, Pa.

Murray: Proceeding of the 5th International Poliornyelitis Conference,Copenhagen, Denmark, July 2628, 1960.

Hillernan ct al.: 'Proc. 88th Annual Meeting Am. Pub. Health A., Nov. 1,1960.

Bulletin of the World Health Organization, vol 22, page 263, 1960.

1. A PROCESS FOR PURIFYING AND CONCENTRATING POLIOMYELITIS VIRALSUBSTANCES FROM POLIOMYELITIS VIRAL SUBSTANCE-CONTAINING FLUIDS,COMPRISING ADDING NUCLEIC ACID TO THE POLIOMYELITIS VIRALSUBSTANCE-CONTAINING FLUID AND ADJUSTING THE PH TO BETWEEN 2-4 ANDRECOVERING THE PRECIPITATED POLIOMYELITIS VIRAL SUBSTANCE.